Kathepsinen en lysosomen

SUMMARY Cathepsin B and cathepsin C (EC 3.4.4.9) are enzymes from bovine spleen which were originally defined by their ability to split certain synthetic substrates of trypsin and chymotrypsin, respectively. Purified preparations of cathepsin B hydrolyze at low pH peptide bonds formed by carboxyl groups of basic amino acids. The activity of cathepsin C at low pH is restricted to the removal of N -terminal dipeptides which must satisfy narrow specificity requirements. Near neutral pH both enzymes can catalyze transamidation reactions. In this thesis it is shown that rat liver and spleen contain enzymes closely resembling cathepsin B and cathepsin C from bovine spleen. The cathepsins B (substrate: benzoyl-Argamide) from both rat tissues and from bovine spleen require high concentrations of cysteine for maximal activation; the enzymes are inhibited by iodoacetamide. Furthermore the enzymes have similar K m values for this substrate and show similar pH optima. Hydrolysis of Gly-Phe -amide by the cathepsins C from rat liver, rat spleen and bovine spleen is also activated by cysteine and inhibited by iodoacetamide. The three enzymes are inhibited by Phe-amide, they have identical Km values for Gly-Phe-amide, they show similar pH-activity curves and catalyze transamidation reactions. The distribution of cathepsin B and that of cathepsin C in rat tissues have been determined. The highest cathepsin B activity was found in the spleen. Cathepsin C activity in the spleen is also considerable, but its activity in the liver is even higher. The distribution of the lysosomal enzymes cathepsinD and acid phosphatase was similar in several respects to the distribution of cathepsin B and of cathepsin C. The cellular localization of the enzymes in the spleen was investigated by total-body X-irradiation, which eliminates the lymphocytes from the spleen (lymphocytes normally make up about 40% of the weight of this organ). After irradiation the cathepsin D content of the spleen was considerably lowered, whereas the activities of the other enzymes per spleen were not significantly altered. From this and other experiments it was concluded that lymphocytes contain rather high concentrations of cathepsin D, but little, if any, cathepsin B, cathepsin C and acid phosphatase. In male and female rats liver and spleen contained comparable activities of cathepsin B, cathepsin C and acid phosphatase. -85After differential centrifl.lgations of spleen homogenates, about 500/0 of the activity of cathepsin C was found in the supernatant fraction. This might be due to rupture of lysosomes from spleen. Differential an isopycnic fractionations, and activation and solubilization experiments indicated that in liver cathepsin B and cathepsin C are located in lysosomes. Acid phosphatase, malic dehydrogenase and glucose -6 -phosphatase were used as reference enzymes for lysosomes, mitochondria and microsomes, respectively. In contrast to some reports in literature no evidence was obtained for the presence of an inhibitor of cathepsin B in the supernatant fraction of liver. From the specificity requirements and the localization of cathepsin B we may conclude that this enzyme may take part in the degradation of material taken up by phagocytosis or pinocytosis. The high degree of specificity of cathepsin C, on the other hand, renders a general hydrolytic activity of this enzyme unlikely in spite of its lysosomal localization. The physiological function of cathepsin C remains, therefore, obscure